Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure

1979 ◽  
Author(s):  
B Wiman
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
High Ionic Strength ◽  
Chromatographic Purification ◽  
Purification Method ◽  
Activity Measurements ◽  
Chromatographic Procedure

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharose and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding ot Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.

Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure

1979 ◽  
Author(s):  
B. Wiman
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
High Ionic Strength ◽  
Chromatographic Purification ◽  
Purification Method ◽  
Activity Measurements ◽  
Chromatographic Procedure

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharase and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS Polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding of Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.

scholarly journals Affinity-chromatographic purification of human α2-antiplasmin

1980 ◽  
Vol 191 (1) ◽  
pp. 229-232 ◽  
Author(s):  
B Wiman
Keyword(s):  
Binding Sites ◽  
Gel Filtration ◽  
Single Step ◽  
Chromatographic Purification ◽  
Purification Method ◽  
Pure Preparation ◽  
A Chain

A new simple and efficient purification method for alpha 2-antiplasmin is described that is based on the interaction between alpha 2-antiplasmin and a fragment from elastase-digested plasminogen constituting the three N-terminal triple-loop structures in the plasmin A-chain (LBSI). After a single-step adsorption of the alpha 2-antiplasmin from plasminogen-depleted plasma to LBSI-Sepharose and elution with 6-aminohexanoic acid, an 80-90% pure preparation with a yield of 50-60% is obtained. The major impurity is fibrinogen, which can easily be removed by gel filtration, and, as a result, a homogeneous fully active alpha 2-antiplasmin preparation is obtained that has the same properties as previously described for alpha 2-antiplasmin. Evidence is put forward that a form of alpha 2-antiplasmin with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma.

HEPARIN INDEPENDENT PURIFICATION OF ANTITHROMBIN III (AT III) BY IMMUNO-AFFINITY CHROMATOGRAPHY RESULTING IN A FUNCTIONALLY INTACT MOLECULE

1987 ◽  
Author(s):  
M Jørgensen
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
Heparin Binding ◽  
Purification Method ◽  
Step Procedure ◽  
At Iii ◽  
Sepharose 4B

Previous methods for purification of AT III are based on its heparin-binding capacity. However, in congenital AT III deficiency abnormal inhibitor molecules with impaired binding of heparin and/or thrombin has been reported. The aim of the present study was to develop a purification method based on immuno-affinity chromatography, and thus independent of the heparin binding capacity.Rabbits were immunized with human AT III purified by a three-step procedure involving dextran sulphate precipitation, affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE-Sephadex A-50. Rabbit immunoglobulins against human AT III were isolated by affinity chromatography using purified human AT III coupled to CNBr-activated Sepharose 4B. Trace amounts of immunoglobulin against human albumin, IgG and IgM were removed by solid phase immunoadsorption. The highly purified immunoglobulins against human AT III were coupled to CNBr-activated Sepharose 4B. This anti-AT III-Sepharose was used for single-step purification of AT III from plasma. Elution was performed by Na-citrate buffer at pH 3.0 and the eluted fractions immediately neutralized. The recovery was more than 60%.The purified AT III appeared as a single protein band in SDS-poly-acrylamide gel electrophoresis with or without reduction. Affinity purified AT III and AT III purified by the three-step procedure were indistinguishable when analyzed by crossed immunoelectrophoresis in the absence and the presence of heparin isoelectrical focusing in polyacrylamid gel at a pH 4-6.5 gradient, and SDS-polyacrylamide gel electrophoresis. AT III antigen concentration was determined by electroimmunoassay and the reactive site concentration determined by titration with purified human thrombin using Phe-Pip-Arg-Nan (S-2238) as substrate. The ratio (active site conc.)/(antigen conc.) was identical in the two AT III preparations. It is concluded that this single-step immuno-affinity chromatography gives a high recovery from plasma of a highly purified functionally intact AT III molecule. The purification method is independent of the heparin binding capacity of AT III. This is of particular importance for the purification and characterization of abnormal AT III molecules with impaired heparin-binding site.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

EXTRACELLULAR CALCIUM AND PLATELET GLYCOPROTEINS

1987 ◽  
Author(s):  
I Jabbal-Gill ◽  
G I Johnston ◽  
S Heptinstall
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Marked Decrease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Monoclonal Antibody ◽  
Alkaline Ph ◽  
Membrane Glycoproteins ◽  
Crossed Immunoelectrophoresis ◽  
The Stability ◽  
Ca Depletion

Platelet membrane glycoproteins lib and Ilia form Ca++-dependent heterodimer complexes that contain binding sites for fibrinogen and therefore are relevant to the ability of platelets to aggregate together. In this study we investigated the effects of extracellular Ca++ on the stability and expression of IIb-IIIa complexes using a IIb-IIIa complex-specific monoclonal antibody M148. Its specificity was examined using crossed immunoelectrophoresis: the antibody reacted only with intact IIb-IIIa complexes and not with either glycoprotein alone.SDS-polyacrylamide gel electrophoresis of immunoprecipitates of soluble glycoproteins that interacted with Ml48 showed that lib and Ilia were present as complexes in Ca++-depleted media at 25°C, pH7.4. However, Ca++-depletion at 37°C, pH7.4 or 37°C, pH8.7 or 25°C, pH8.7 caused dissociation of the complexThe effect of extracellular Ca++ on the expression of IIb-Illa complexes on the surface of intact platelets was studied by a technique which is based upon indirect binding of M148 using a fluorescent- labelled second antibody (FITC-RAM) and measuring the fluorescence per platelet using the FACS IV cytofluorometer. Intact platelets were incubated in Ca++-depleted media at 25°C, pH7.4 or 37°C, pH7.4 either (i) prior to or (ii) after adding M148. At 25°C increased M148-binding was observed, compared to the value prior to Ca++-depletion. This increased binding could be reversed by adding Ca++ back to the preparation. Under condition (i) at 37°C a marked decrease in M148 binding was observed, which could not be reversed by restoring Ca++, while under condition (ii) at 37°C the results were the same as at 25°C.Our studies demonstrate that (a) Ca++-depletion at 37°C and/or alkaline pH causes dissociation of the Ilb-IIIa complex (b) Ca++ depletion at 25°C possibly alters distribution of the complexes thereby increasing their availability to the antibody and (c) M148 prevents the dissociation of complexes in Ca++-depleted media at 37°C, possibly by holding lib and Ilia together

scholarly journals Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield

1981 ◽  
Vol 197 (2) ◽  
pp. 519-522 ◽  
Author(s):  
E G Afting ◽  
M L Recker
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Acid Proteinase ◽  
Chromatographic Purification

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

scholarly journals The distribution of iron between the metal-binding sites of transferrin human serum

1980 ◽  
Vol 185 (2) ◽  
pp. 483-488 ◽  
Author(s):  
J Williams ◽  
K Moreton
Keyword(s):  
Human Serum ◽  
Metal Binding ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Iron Binding ◽  
Metal Binding Sites ◽  
Fresh Serum ◽  
Marked Preference ◽  
Terminal Site

The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250-256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at −15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction.

scholarly journals Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement

1986 ◽  
Vol 233 (3) ◽  
pp. 819-825 ◽  
Author(s):  
J Janatova
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Gamma Chain ◽  
Complement Components ◽  
The Third ◽  
Disulphide Bonds ◽  
Reduced State

Disulphide bonds contribute significantly to the maintenance of structural/functional integrity of many proteins. Therefore it was of interest to study the distribution and the effect of disulphides on conformation of complement components C3 and C4. These proteins are precursors of several fragments with various binding sites and distinct physiological functions. The constituents of C3c (beta, alpha 27, alpha 43) and those of C4c (beta, alpha 27, alpha 16, gamma) were investigated, since other fragments of C3 or C4 do not participate in interchain linkages. Inter-and intra-chain disulphide bonds in C3c and C4c were localized by using a modification of conventional SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis such that the change in mobility of disulphide-bond-containing proteins can be detected throughout the transition from a non-reduced to a fully reduced state. Several forms of the alpha 43 fragment from C3, and of the gamma-chain of C4, with different mobilities can exist, depending on the number of intra-chain disulphide bonds reduced. The intermediates (heterodimers) generated by a partial reduction of C3c or C4c were characterized by two-dimensional SDS/polyacrylamide-gel electrophoresis performed in the absence, then in the presence, of beta-mercaptoethanol. The inter-chain linkages in C3c were determined to be beta-alpha 27 and alpha 27- alpha 43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are beta-alpha 27 and alpha 16-gamma. The third interchain bond in C4 (alpha 27-gamma, tentative) remains to be determined.

scholarly journals The effect of salt concentration on the iron-binding properties of human transferrin

1982 ◽  
Vol 201 (3) ◽  
pp. 527-532 ◽  
Author(s):  
J Williams ◽  
N D Chasteen ◽  
K Moreton
Keyword(s):  
Salt Concentration ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Iron Binding ◽  
Iron Release ◽  
Equilibrium Conditions ◽  
Binding Properties ◽  
Kinetics Of ◽  
Terminal Site

The salt dependence of the iron-binding properties of transferrin was studied by urea/polyacrylamide-gel electrophoresis. The distribution of iron between the N-terminal and C-terminal binding sites under equilibrium conditions and the rates of release of iron from the two sites were studied. It was found that salt increases the thermodynamic stability of iron binding in the N-terminal site relative to the C-terminal site. Similar behaviour is observed for the kinetics of iron release, where salt retards the rate of removal of iron from the N-terminal site but facilitates removal from the C-terminal site.

scholarly journals Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin

1983 ◽  
Vol 213 (2) ◽  
pp. 345-353 ◽  
Author(s):  
A Danielsson ◽  
I Björk
Keyword(s):  
Rate Constants ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dissociation Rate ◽  
Exchange Chromatography ◽  
Stable Complex ◽  
Intermediate Step ◽  
First Order ◽  
Activity Measurements ◽  
Dissociation Rate Constants

Purification of antithrombin-thrombin complex by ion-exchange chromatography on DEAE-agarose resulted in predominantly monomeric complex, whereas purification on matrix-linked heparin produced large amounts of aggregated complex. Monomeric antithrombin-thrombin complexes formed in the presence and in the absence of heparin had similar conformations and heparin affinities. Moreover, the first-order dissociation rate constants, measured by thrombin release, of these complexes were similar, 2.3 × 10(-6)-3.4 × 10(-6)S-1, regardless of whether newly formed or purified complex was analysed. Similar dissociation rate constants were also obtained for purified complex formed with or without heparin, from analyses by dodecyl sulphate/polyacrylamide-gel electrophoresis of the release of modified antithrombin, cleaved at the reactive-site bond. No dissociation of intact antithrombin from the complex was detected by activity measurements or by gel electrophoresis. Aggregation of the complex was found to be accompanied by a decrease in apparent dissociation rate. The similar properties of antithrombin-thrombin complexes formed with or without heparin support the concept of a catalytic role for the polysaccharide in the antithrombin-thrombin reaction. Furthermore, the results indicate that the reaction between enzyme and inhibitor involves the rapid formation of an irreversible, kinetically stable, complex that dissociates into active thrombin and modified, inactive, antithrombin by a first-order process with a half-life of about 3 days. The inhibition thus resembles a normal proteolytic reaction, one intermediate step of which is very slow.

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